Immunological relationships among proteins making up the Bacillus thuringiensis subsp. israelensis crystalline toxin

Abstract

The immunological relationships among the proteins of the mosquito larvicidal toxin produced by Bacillus thuringiensis subsp. israelensis have been investigated by using polyclonal antisera specific for the 28-, 70-, and 135-kilodalton proteins. Each of these proteins was immunologically distinct. There was no cross-reaction among the three proteins and the two non-homologous antisera. Treatment of toxin proteins with larval gut enzymes for 20 h identified protease-resistant domains at approximately 65, 38, and 22 kilodaltons. Similar domains were generated by treatment with trypsin and chymotrypsin. Our immunological and kinetic data indicate that the 28-kilodalton protein is degraded successively to protein bands at 26, 25, 23, and 22 kilodaltons, the 70-kilodalton protein is degraded to a protein at 38 kilodaltons, and the 135-kilodalton protein is degraded successively to protein bands at 94, 72, and, probably, 65 kilodaltons. Solubilized toxin possesses two biological activities, larvicidal and general cytolytic (hemolytic). We used nondenaturing gel electrophoresis to show that the hemolytic activity resides in the 28-kilodalton protein. However, higher-molecular-weight proteins are required to achieve the level of toxicity observed in intact toxin.

Toxicity of protease-resistant domains from the delta-endotoxin of Bacillus thuringiensis subsp. israelensis in Culex quinquefasciatus and Aedes aegypti bioassays

“…No spontaneous breakdown products were observed; controls without added proteases maintained the same protein profiles as fresh intact crystals, even when incubated at 22 to 25°C for 24 h. The presence of EDTA during solubilization is necessary to inactivate contaminating metalloproteases (20). The sizes of these protease-resistant domains were the same as those we reported previously (18). Furthermore, no undigested 135-to 140kilodalton proteins were observed following chymotrypsin digestion, even with the more sensitive silver stain.…”

“…It is composed of multiple protein subunits and has both larvicidal activity and general cytolytic activity (3,13,20). A 28-kilodalton protein is responsible for the general cytolytic activity (2,10,18), but larvicidal activity is due to a synergistic combination of the 28-kilodalton protein and greater-molecular-mass proteins (18,23,27). The 28-, 70-, and 135-kilodalton proteins of the crystal are immunologically distinct and contain proteaseresistant domains of 22, 38, and 65 kilodaltons, respectively (18).…”

Mosquitocidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes.

“…The 27-kDa' CytA protein is hemolytic and has no or marginal mosquitocidal activity (see reference 58 and references therein). In contrast, the 134-, 128-, and 70-kDa proteins are not hemolytic, and individually they show significant toxicity to Aedes mosquito larvae (38,57,95,152,160). This has been confirmed with the individual recombinant proteins expressed in heterologous cells which are free of all other toxins.…”

BACILLUS THURINGIENSIS USE IN AGRICULTURE: A MOLECULAR PERSPECTIVE

“…ovoid complex resides in the protease-resistant domains of three distinct proteins (i.e. the final products of proteolytic degradation) : approximately 25, 30-35 and 53-67 kDa, derived from protoxins of the following molecular weights: 28, 65-70 and 130 kDa (Sekar & Carlton, 1985;Pfannenstiel et al, 1986;Chilcott & Ellar, 1988; Delecluse et al ., 1988;Porter et al, 1993). T h e activated form of the CryIV D toxin (a c. 39 kDa fragment) is highly active against Aedes aegypti L. larvae, whereas for the other CryIV proteins, solubilization greatly (50-1 oo-fold) reduces toxicity (Chilcott & .…”